Process for the manufacture of degarelix and its intermediates

ABSTRACT

The present invention provides a manufacturing process for preparing a peptide, preferably a decapeptide, such as degarelix, by incorporating p-nitro-phenylalanin in the amino acid sequence preferably during stepwise solid phase synthesis, and converting these into the required amino acids Aph(Hor) and/or D-Aph(Cbm), preferably while attached to a solid phase. The invention further provides intermediates useful in the manufacturing process.

FIELD OF THE INVENTION

The present invention relates to a process, or method of synthesis, forpreparing peptides wherein at least one amino acid in the peptide ischaracterized as an unnatural amino-phenylalanin derivative, specifiedas Aph(Hor) or Aph(Cbm). In particular the invention relates to thesynthesis of decapeptides, such as degarelix, and its protectedprecursor, and other useful intermediates. Intermediates useful in themanufacturing process are also described. The method of synthesis ischaracterized by the use of p-nitro-phenylalanine, which is incorporatedin the growing peptide chain at the respective positions of Aph(Hor)and/or D-Aph(Cbm) in the final peptide sequence. Another method ofsynthesis is based on the use of a compound of Formula II, which isitself synthesized by the use of p-nitro-phenylalanine.

BACKGROUND OF THE INVENTION

The synthesis of peptides carrying at least one amino-phenylalaninderivative, such as for example Aph(Hor), Aph(Cbm) or Aph (Atz) in theiramino acid sequence is challenging. The synthesis often results in aproduct with a high amount of impurities (such as deletion products orproducts of side reactions).

WO99/26964 is addressing the problem of synthesizing azaline B, adecapeptide that carries two identical unnatural amino acids Aph(Atz) atpositions 5 and 6. The suggested synthesis starts with synthesizing ofthe central “5/6” dipeptide fragment. The core of this method is thesimultaneous incorporation of both aminotriazole groups(Atz=5-(3′-amino-1H-1′,2′,4′-triazolyl). First, a nitro-substitutedPhe-Phe dipeptide is generated. The nitro groups on the dipeptide arethen reduced to obtain the amino substituted dipeptide. The1,2,4-triazole groups are then formed simultaneously on theamino-substituted phenylalanine groups by reacting the reduced dipeptidefirst with diphenyl cyanocarbonimidate and then with hydrazine. Thefinal “5/6” dipeptide fragment is obtained by reaction with a suitablebase and then reacted with the missing fragments in a liquid reaction.Obviously this elegant approach is not suitable for the synthesis ofdecapeptides with different Aph side chains, such as degarelix.Furthermore, it is not possible to be performed as solid state peptidesynthesis, but as liquid phase synthesis only. In WO99/26964 the solidstate synthesis is regarded unsuitable for providing an improvedsynthesis of degarelix (page 7).

It remains a challenging task to efficiently synthesize peptides withdifferent derivatives of Aph in their 5 and 6 position.

U.S. Pat. No. 5,925,730 discloses a selection of decapeptides, whichhave GnRH antagonistic properties and may be used for treatment. All ofthese antagonists have a derivative of Aph in the 5- and/or 6-positions.They are depicted by the generic sequenceX-D-2Nal-(A)D-Phe-D-3Pal-Ser-Xaa5-Xaa6-Leu-Xaa8-Pro-Xaa10, wherein Xaa5is 4Aph(Q1) or 4Amf(Q1) with Q1 being D- or L-Hor or D- or L-Imz, andXaa6 is D-4Aph(Q2), D-4Amf(Q2), with Q2 being For, Ac, 3-amino-1,2,4triazole, or Q1. Alternatively, when Xaa6 contains Q, wherein Q is

or D- or L-Hor or D- or L-Imz, Xaa5 may have Ac, For or3-amino-1,2,4-triazole as Q1.

The Aph derivative may contain a carbamoyl group or a heterocycleincluding a urea in its side chain, for exampleAc-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(hydroorotyl)-D-4Aph(acetyl)-Leu-Lys(isopropyl)-Pro-D-Ala-NH2,andAc-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(hydro-orotyl)-D-4Amf(Q2)-Leu-Lys(isopropyl)-Pro-D-Ala-NH2,wherein Q2 is Cbm or MeCbm. The most prominent example of such a peptideis the decapeptide degarelix. Degarelix, is an approved medicinalproduct.

In the following an improved synthesis of peptides with Aph derivatives,such as for example Aph(Hor) or Aph(Cbm), in their sequence, isdisclosed that results in a product of high yield and purity without theneed to invest in the production or procurement of specialty aminoacids, such as Aph(Hor) or Aph(Cbm). A synthesis for degarelix is alsodisclosed.

The compoundAc-D-Nal-D-Cpa-D-Pal-Ser-Aph(Hor)-D-Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH₂,also known as degarelix is represented by the Formula (I), the numbersindicate the direction of counting the relevant amino acid positions:

Degarelix (a pharmaceutical composition thereof is marketed under thetrade name Firmagon®), also known as FE200486 is a synthetic lineardecapeptide and a third generation gonadotropin releasing hormone (GnRH)receptor antagonist (a GnRH blocker). It was approved by the U.S. Foodand Drug Administration (FDA) on 24 Dec. 2008 for treatment of patientswith advanced prostate cancer. It acts by decreasing the level ofluteinizing hormone and suppressing testosterone and prostate-specificantigen (PSA) secretion (G. Jiang et al. J. Med. Chem. 44, 2001,453-467; M. P. Samant et al. Bioorg Med. Chem. Lett. 15, 2005,2894-2897). It has a number of advantages over many other GnRHantagonists, such as high affinity and potency at the GnRH receptor,water solubility sufficient for the development of an injectableformulation, as well as properties enabling the development of asustained release form (M. Steinberg. Clin. Ther. 31, 2009, 2312-2331),which allows to decrease testosterone concentration fast and to maintainthis level for an increased amount of time.

Degarelix, as represented by formula I was first reported ininternational publication no. WO 98/46634. WO 98/46634 describes thesynthesis of degarelix using solid phase peptide synthesis (SPPS) onMBHA resin and Boc-Na protection of amino acids. The removal ofBoc-protective group after each coupling step was achieved by 50%solution of trifluoroacetic acid in dichloromethane with addition of 1%of m-cresole. D-Aph(Cbm) and L-Aph(Hor) residues were introduced in thepeptide structure as D-Aph(Fmoc) and L-Aph(Fmoc) followed by consecutiveFmoc-deprotection by applying a 25% solution of piperidine indimethylformamide and treatment with t-butyl isocyanate and hydrooroticacid respectively. The completion of SPPS and cleavage of the peptidewith hydrogen fluoride resulted in obtaining full-length degarelix.Alternatively, the Fmoc-protective group of Aph in position 5 wasremoved only after the completion of solid phase synthesis and thepeptide eventually modified with L-dihydroorotic acid.

The above mentioned synthetic processes do not result in degarelix ofhigh purity. The processes involve use of hazardous reagents such ashydrogen fluoride. Moreover, the final cleavage of the peptide undervery strong acidic conditions induces a partial degradation of thepeptide, which causes a significant amount of yield to be lost.Furthermore, the impurity profile achieved by these syntheses requirespurification steps which result in higher losses.

In the following years alternative methods suitable for the industrialproduction of degarelix, several approaches based on SPPS and LPPS, aswell as the combination of these methods, were proposed.

WO2010/121835 discloses a method of stepwise Fmoc based SPPS synthesisof full-length degarelix using the two commercially available p-aminophenylalanine derivatives having a carbamoyl moiety and a dihydroorotylmoiety, Aph(Cbm) and Aph(Hor), respectively. These residues wereintroduced without any modification during a regular SPPS, whichcomprised only sequential coupling and deprotection steps. Degarelixobtained by this method showed to contain the4-([2-(5-hydantoyl)]acetylamino)-phenylalanine analog of degarelix,which is known to be formed after dihydroorotic moiety isomerisation inbasic conditions, in an amount lower than 0.3%.

Another method based on Fmoc-SPPS is described in WO2011/066386, wherethe trityl protective group for serine is used, instead of the t-butyl.The use of this more acid-labile protective group allowed to decreasethe time of the final cleavage and to get a crude product with a loweramount of impurities. WO2011/066386 also discloses the synthesis ofdegarelix via a (9+1) condensation strategy and (3+6+1) condensationstrategy.

Similarly, a liquid phase condensation process for degarelix preparationwas disclosed in WO2012/055903. Various fragments of degarelix weresynthesized in solution or on the solid phase and coupled in solution.In particular, [4+6] and [3+7] strategies were described.

Chinese patent application, CN103992392, discloses a method for thesynthesis of degarelix by the introduction of Aph(Hor) wherein thebase-labile protection group Dde for the p-amino group was used. Itsdeprotection with 2% hydrazine in dimethylformamide with the followingcoupling of hydroorotic acid on the solid phase and cleavage from theresin yielded the final crude product.

Chinese patent application, CN102952174, discloses a method for thesynthesis of degarelix on the Rink amide resin, wherein Aph(Trt) wasintroduced during Fmoc-based SPPS. The trityl protection was removedwith 10% trifluoroacetic acid. The unprotected amino group was coupledwith hydroorotic acid.

All the above mentioned processes include the insertion of Aphderivatives into the structure of degarelix that have to be prepared andpurified previously by standard methods of organic chemistry. Itcomplicates the overall synthesis of the peptide. Moreover, theintroduction of these bulky residues in the peptide sequence, preferablyduring a step-by-step solid phase synthesis, is complicated because oftheir steric hindrance and, therefore may cause difficulties in thecoupling steps. These residues (p-amino phenylalanine derivativesAph(Hor) and D-Aph(Cbm) in particular in the 5^(th) and 6^(th) positionof the decapeptide respectively), can also induce the aggregation of thegrowing peptide chain due to inter- and intramolecular hydrogen bondformation. All these factors can lead to a noticeable formation of sideproducts, such as, for example, deletion products, i.e. side productsformed which are lacking an amino acid of the intended sequence.

The methods known for the preparation of degarelix suffer from one ormore of the following drawbacks: low yields obtained due to theformation of side products (deletion products), and unfavorable impurityprofiles leading in more losses during purification of the crudepeptide, and the need to use hazardous reagents and complicatedprocesses.

Thus, there remains a need to develop an efficient, simple andindustrially viable synthetic process which can overcome the drawbacksof the prior art and which provides (crude) peptides, in particulardegarelix, in high yield and a favorable impurity profile, facilitatingthe generation of purified peptides, such as degarelix with high yieldand high purity.

Object of the Invention

It is an objective of the present invention to overcome theabove-mentioned drawbacks of the prior art.

It is another objective of the present invention to provide an improvedprocess for the synthesis of the decapeptide degarelix or apharmaceutically acceptable salt thereof without the use of hazardousreagents.

It is another objective of the present invention to provide an improvedprocess for the synthesis of degarelix or a pharmaceutically acceptablesalt thereof, which results in crude degarelix in a high yield and animpurity profile facilitating a simplified purification process and/or asignificantly higher yield and purity than achieved in the state of theart.

It is a further objective of the present invention to provide usefulintermediates for the synthesis of degarelix or a pharmaceuticallyacceptable salt thereof.

SUMMARY OF THE INVENTION

The present invention provides an improved process for the preparationof peptides, or pharmaceutically acceptable salts thereof, wherein thepeptide is characterized as being a peptide with an Aph(Hor) and/or anAph(Cbm) in the peptide sequence. Preferably the Aph(Hor) and anAph(Cbm) are the 5^(th) and/or 6^(th) position. Preferably the peptideis a decapeptide. The most preferred peptide is the decapeptidedegarelix, with an Aph(Hor) and an Aph(Cbm) in the 5^(th) and 6^(th)position, respectively. The process of the present invention results ina crude peptide of higher purity, with high yield, and an improvedimpurity profile.

The method of synthesis for preparing a peptide, which is characterizedas having at least one of, the amino acid Aph(Hor), preferably in the5^(th) position, or the amino acid Aph(Cbm), preferably in the 6^(th)position, in its sequence, is characterized by the incorporation of atleast one p-nitrophenylalanine into the growing peptide chain. Themethod preferably comprises the use of the amino acidpara-nitrophenylalanine or a nitro-peptide, which is characterized ascomprising one or two p-nitrophenylalanine residues. In preferredembodiments these nitro-peptides are the compounds of formula II,formula V, and/or formula VII. The method is further characterized bythe steps which subsequently transform the incorporatedp-nitrophenylalanin into either Aph(Hor) or Aph(Cbm), as required by thefinal target peptide sequence. In a another aspect, a synthesis forpreparing degarelix of formula I,

or a pharmaceutically acceptable salt thereof, is provided, wherein themethod comprises the use of either the amino acid p-nitrophenylalanine,or a nitro-peptide, which is characterized as comprising one or twop-nitrophenylalanine residues. In preferred embodiments thesenitro-peptides are the compounds of formula II, formula V, and formulaVII.

A p-nitro-phenylalanine may be incorporated for either the 5^(th) or the6^(th) position of degarelix or both, the p-nitrophenylalanineincorporated in the 6^(th) position within the degarelix is the Disomeric form, and the p-nitrophenylalanine incorporated in the 5^(th)position within the degarelix is the L isomeric form. When L or D is notspecifically indicated the L form is referred to.

This may be achieved by introducing p-nitro-phenylalanine instead of thebulkier phenylalanine derivatives known from the art into a precursorpeptide, for example by adding a p-nitro-phenylalanine to Leu-Lys(iPr,BOC)-Pro-D-Ala, or to a compound of Formula XI, or toD-Aph(Cbm-tBu)-Leu-Lys(iPr, BOC)-Pro-D-Ala, or to a compound of formulaIX. The resulting intermediates may be used to produce degarelix, forexample by consecutive reduction to p-amino-phenylalanine andmodification thereof.

Due to the difference of the side groups, both Aph amino acid sidegroups cannot be built up simultaneously,as is possible for azaline B,but instead for example the Aph(Cbm) has to be built first and then hasto be protected with a suitable protective group, before the secondamino acid side group, for example Aph(Hor) is built up by reduction ofthe inserted nitro group. One challenge is therefore, to find suitableconditions for these reduction reactions. The inventors found thatstannous chloride (Sn(II)Cl₂ did work best, under the suitableconditions as indicated below. Thereby they developed an improvedsynthesis for degarelix.

Preferably the syntheses described herein are performed as solid phasepeptide syntheses. Therefore the schemes will show the peptide bound toa “Resin”. It is therefore preferred that the “Resin” is a solidsupport. In principal the same reaction scheme may be performed wherein“Resin” is H, when the synthesis steps are performed in solution.

In solid phase synthesis the protection may be based on the so calledBOC protection strategy or the Fmoc protection strategy. More preferablythe synthesis is based on the Fmoc protection strategy, wherein theterminal protection group Pg is a base-labile protective group,preferably Fmoc. It is most preferred that all solid state synthesesaccording to the invention are performed with the Fmoc protectionstrategy.

Another challenge for such syntheses is the capability to be scaled-up.Usually solid phase syntheses are not ideally suited for scale up.Surprisingly the synthetic approaches described below could be scaled upto a final production yield of at least 0.5 g.

In another aspect, a peptide synthesis for preparing degarelix offormula I, or a pharmaceutically acceptable salt thereof, is providedcomprising converting a compound of formula II,

-   -   wherein Pg is a terminal protecting group, X is a side chain        protecting group and Y is a side chain protecting group or H,        and Resin is a solid support;

into degarelix or a pharmaceutically acceptable salt thereof.

Preferably this synthesis, as well as the synthesis of other peptidesaccording to the invention, is performed as a solid phase peptidesynthesis. Even more preferably this synthesis is carried out with theFmoc scheme, wherein Pg is Fmoc.

In another aspect, a process of converting the compound of formula IIinto degarelix or a pharmaceutically acceptable salt thereof isprovided, characterized as comprising the steps of:

-   -   a) treating the compound of formula II with a reducing agent to        form a compound of formula III,

-   -   b) reacting the compound of formula III with an activated        dihydroorotic acid, to form a compound of formula IV,

-   -   c) repeating the following steps (i)-(ii) until formation of a        protected decapeptide, preferably attached to a resin by solid        phase peptide synthesis:        -   (i) deprotecting the protected peptide attached to the resin            to remove terminal protecting group,        -   (ii) coupling of the protected amino acid (according to            required sequence) to the terminal amino group residue            attached to the resin using a coupling reagent to form a            protected peptide attached to the resin,    -   d) deprotecting the protected decapeptide attached to resin to        remove the terminal protecting group,    -   e) acetylating the N-terminus of the resulting decapeptide in        the presence of an acetylating agent, and    -   f) cleaving the acetylated decapeptide from the resin to form        degarelix or a pharmaceutically acceptable salt thereof

wherein Pg is a terminal protecting group, X is a side chain protectinggroup and Y is a side chain protecting group or H, and Resin is a solidsupport.

In another aspect, a process of converting the compound of formula IIinto degarelix or a pharmaceutically acceptable salt thereof, preferablyas a solid phase peptide synthesis, is provided characterized ascomprising the steps of:

-   -   a) providing the compound of formula II,    -   b) repeating the following steps (i)-(ii) until the formation of        a protected decapeptide attached to the resin:        -   (i) deprotecting the protected peptide attached to the resin            to remove the terminal protecting group,        -   (ii) coupling of the protected amino acid (according to            required sequence) to the terminal amino group residue            attached to the resin using a coupling reagent to form a            protected peptide attached to the resin,    -   c) deprotecting the protected decapeptide attached to the resin        to remove terminal protecting group,    -   d) acetylating the N-terminus of resulting decapeptide in the        presence of acetylating agent to give a compound of formula V,

-   -   e) treating the compound of formula V with a reducing agent to        form a compound of formula VI,

-   -   f) reacting the compound of formula VI with an activated        dihydroorotic acid, to form a protected decapeptide attached to        the resin; and    -   g) cleaving the decapeptide from the resin to form degarelix or        a pharmaceutically acceptable salt thereof        -   wherein X, and Z are side chain protecting groups, and Y is            a side chain protecting group or H, and “Resin” is a solid            support.

As another aspect a process for the solid phase synthesis of thecompound of formula II, is provided, comprising the steps of:

-   -   a) reducing a compound of formula VII,

-   -   wherein Pg is a terminal protecting group, X is a side chain        protecting group, and “Resin” is a solid support        with a reducing agent to form a compound of formula VIII,

-   -   wherein Pg is a terminal protecting group; X is a side chain        protecting group and “Resin” is a solid support    -   b) reacting the compound of formula VIII with alkyl isocyanate        or alkali metal cyanate to form a compound of formula IX,

wherein Pg is a terminal protecting group, X and Y are side chainprotecting groups or, Y is H if an alkali metal cyanate is used, andresin is a solid support, and

-   -   c) deprotecting the compound of formula IX to remove the        terminal protecting group followed by coupling with the        protected amino acid of formula X,

-   -   -   wherein Pg is a terminal protecting group

in the presence of a coupling reagent to form the compound of formulaII.

As another aspect a process for the solid phase synthesis of thecompound of formula VII, is provided, comprising the step ofdeprotecting the compound of formula XI,

-   -   wherein Pg is a terminal protecting group, X is a side chain        protecting group, and resin is a solid support,

to remove the terminal protecting group followed by coupling withprotected amino acid of formula X (D-isomer) in the presence of acoupling reagent to form the compound of formula VII.

Another aspect of the present invention is to provide the compounds offormula II, formula V and formula VII.

Still another aspect of the present invention is the use of thesecompounds in the synthesis of degarelix.

DESCRIPTION OF FIGURES

FIG. 1: This figure shows a scheme of the solid phase synthesis ofdegarelix acetate using a suitable peptide precursor therebyincorporating L-p-nitro-Phe and D-p-nitro-Phe residues at the 5^(th) and6^(th) position, respectively.

FIG. 2: This figure shows a scheme of the solid phase synthesis ofdegarelix acetate by using a suitable peptide precursor therebyincorporating L-p-nitro-Phe residue at 5^(th) position.

FIG. 3: This figure illustrates the HPLC profile of the pentapeptideafter the reduction step with dithionite, according to example 3.1.

FIG. 4: This figure illustrates the HPLC profile of the pentapeptideafter the reduction step with tin chloride, according to example 3.2.

DETAILED DESCRIPTION OF THE INVENTION

The term “terminal protecting group” as used herein refers to aprotecting group of the carbamate type. The preferred terminalprotecting group is Fmoc (9-fluorenylmethyloxycarbonyl), which can beremoved in acidic conditions. In another embodiment the terminalprotecting group is BOC.

As used herein, the term “side-chain protecting group” is referring to aprotecting group for an amino- or hydroxyl group, in a side chain of thepeptide, which is removed under suitable conditions, it is not removedunder those conditions when the terminal protecting group or anotheramino-protecting group is removed. Preferably side chain protectinggroups are included to protect side chains of amino acids which areparticularly reactive or labile, to avoid side reactions and/orbranching of the growing molecule. The criterion for selecting asuitable side chain protecting group X, Y and Z is that the protectinggroup should generally be stable to the reagent under the reactionconditions selected for removing the terminal amino protecting group ateach step of the synthesis and should be removable upon completion ofthe synthesis of the desired amino acid sequence under reactionconditions that will not alter the peptide chain. Preferably X is anamine protecting group such as tertbutyloxycarbonyl(Boc); Y is ahydrogen or an amine protecting group such as an alkyl or morepreferably a tert-butyl group; Z is a hydroxyl protecting group such astert-butyl.

As used herein the term “salt” includes acid salts, such as for example,hydrochlorides, and acetates.

As used herein the term “peptide” is understood to be a sequence ofamino acids, or amino acid derivatives, coupled one to another bypeptide bonds, of a minimum length of 2 amino acids. The term“nitro-peptide” as used herein is such a peptide comprising one or twop-nitro-phenylalanine residues.

The term decapeptide is comprised of a chain of ten amino acids coupledby peptide bonds.

Throughout the description wherein the term “Resin” is used it isunderstood to comprise a solid support suitable to perform solid phasepeptide synthesis, which allows obtaining a C-terminal amide aftercleavage from the resin, unless “Resin” is explicitly declared to be H(proton). In that case the free (unbound) form of the peptide iscomprised in the reference. A preferred solid support for a Fmoc basedsynthesis is one selected from the group consisting of the Rink Amideresin, Rink amide AM resin and Rink amide MBHA resin; more preferablythe resin is the Rink amide MBHA resin. A preferred solid support for aBoc based synthesis is Benzhydrylamine resin (BHA) or4-Methylbenzhydrylamine resin (MBHA), more preferablyMethylbenzhydrylamine resin.

As used herein the term “activated acid” means an acid derivative thatis able to form a peptide bond after the reaction with an amine.

The term “activated dihydroorotic acid” is understood to bedihydroorotic acid activated by the presence of one or more couplingreagents, or to be a derivative of dihydroorotic acid, such asHOR—CO—Hal, where Hal is Cl, Br or F.

The present invention provides an improved process for the preparationof a peptide, preferably a peptide, characterized as having at least oneAph derivative in its sequence, preferably an Aph(Hor) or Aph(Cbm), morepreferably a decapeptide or a pharmaceutically acceptable salt thereofby incorporation of p-nitrophenylalanine residues in those positionswhich are Aph derivatives, preferably Aph(Hor) and/or Aph(Cbm), in thefinal peptide. Preferably the peptide is a decapeptide with Aph(Hor) inthe 5^(th) and Aph(Cbm) in the 6th position. Most preferably thedecapeptide is degarelix. The nitro groups of these p-nitrophenylalanineresidues are subsequently reduced to amino groups and these aresubsequently modified to obtain Aph(Hor) and D-Aph(Cbm), preferably inposition 5 and 6 of the decapeptide, respectively. The modification ofthese does take place sequentially (not simultaneously).

The inventors have found that surprisingly pharmaceutically puredegarelix can be manufactured by introduction of p-nitrophenylalanineinstead of their bulkier derivatives, which are then transferred intothe correct amino acid derivatives, Aph(Hor) and D-Aph(Cbm) group, whilebeing incorporated in the peptide, preferably while being bound to asolid phase (i.e. the resin) during SPPS. They also found that thisstrategy noticeably facilitates or simplifies peptide chain formation.Unexpectedly, this degarelix synthesis characterized by the use ofcorresponding p-nitro precursors, yields the crude product with a highpurity and a beneficial impurity profile, in comparison to the classicalapproach, disclosed in WO2010/121835, facilitating a much betterseparation during HPLC purification and thus increasing the final yield.

In a first aspect, a peptide synthesis for preparing degarelix or apharmaceutically acceptable salt thereof is provided. The improvedpeptide synthesis of degarelix or a pharmaceutically acceptable saltthereof, is based on the strategy of incorporating into the sequence thep-nitro phenylalanine residues at the 5^(th) position in the amino acidsequence used to synthesize degarelix, with subsequent reduction of thenitro groups to amino groups and their modification to obtain Aph(Hor)before further continuing with the synthesis. In a preferred embodimentthe synthesis is a solid-phase peptide synthesis. A preferred syntheticapproach comprises converting the compound of formula II into degarelixor a pharmaceutically acceptable salt thereof.

The new compound of formula II can be converted to degarelix or apharmaceutically acceptable salt thereof by different procedures. Two ofthese are described herein. In one embodiment of the invention, thepeptide synthesis of degarelix or a pharmaceutically acceptable saltthereof, involving the compound of formula II, is based on the strategyof subsequent reduction of the incorporated nitro group to an aminogroup and its modification to obtain Aph(Hor) before further continuingwith the solid phase synthesis.

The process comprises the steps of:

-   -   a) treating the compound of formula II with a reducing agent to        form the compound of formula III,    -   b) reacting the compound of formula III with an activated        dihydroorotic acid, which may be dihydroorotic acid activated by        the presence of coupling reagent, to form the compound of        formula IV,    -   c) repeating the following steps (i)-(ii) with different amino        acids until the protected decapeptide, which is attached to a        resin, is formed:        -   (i) deprotecting the protected peptide attached to the resin            to remove the terminal protecting group,        -   (ii) coupling of the protected amino acid (according to            required sequence) to the terminal amino group residue            attached to the resin using a coupling reagent to form the            protected peptide attached to the resin,    -   d) deprotecting the protected decapeptide attached to resin to        remove the terminal protecting group,    -   e) acetylating the N-terminus of the resulting decapeptide in        the presence of acetylating agent; and    -   f) cleaving the acetylated decapeptide from the resin to achieve        a decapeptide with an Aph(Hor) amino acid at position 5, or a        pharmaceutically acceptable salt thereof. Preferably this        decapeptide is degarelix.

The reducing agent used in step a) is selected from, but not limited to,sodium dithionite, tin (II) chloride or iron powder. Preferably, thereduction is carried out in the presence of tin (II) chloride.

In step b) the compound of formula III is reacted with an activateddihydroorotic acid to form the compound of formula IV. When, theactivation is facilitated by the presence of a coupling reagent,suitable coupling reagents are N,N′-diisopropylcarbodiimide,dicyclohexyl-carbodiimide, ethyl-dimethylaminopropyl carbodiimide (EDC).Preferably, the reaction is carried out in the presence ofN,N′-diisopropylcarbodiimide.

The reaction may also be carried out in the presence of a couplingreagent and an additive. The addition of an additive has been found toreduce racemisation during the formation of amide linkage (i.e. peptidebond formation). An additive, when added to the coupling reactionresults in an increased yield and reduced racemisation rates of thepeptides formed. Additives form activated esters with amino acids. Theysuppress the side-reaction, such as formation of oxazolone andN-acylurea. A suitable additive is preferably selected from the groupcomprising of 1-hydroxybenzotriazole (HOBt), 2-hydroxypyridine N-oxide,N-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole andendo-N-hydroxy-5-norbornene-2,3-dicarboxamide. More preferably, thereaction is carried out in the presence of N,N′-diisopropylcarbodiimideand 1-hydroxybenzotriazole.

In step c), the protected decapeptide, which is still attached to theresin is prepared by extension of the peptide chain, the concept isknown as solid phase peptide synthesis, which involves first removingthe terminal protecting group from the protected peptide attached to theresin and then coupling with terminally protected amino acids stepwisein the desired order until the desired decapeptide is formed.

In a first step (i), the N-terminal protecting group—if it is Fmoc—canbe removed by treatment with a base, preferably an organic base, morepreferably an amine. The base may be selected from the group consistingof piperidine, piperazine, DBU and diethylamine, preferably piperidineand DBU, and most preferably, piperidine are used.

In a second step (ii), coupling of the protected peptide bound to theresin with the next protected amino acid is carried out in the presenceof a coupling reagent selected from the group comprising of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide,2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), or any coupling reagents on the base of uronium or phosphoniumsalts. Preferably, the reaction is carried out in the presence of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate.

The coupling reaction may be carried out in the presence of a baseselected from the group comprising of tertiary amine likediisopropylethylamine, triethylamine, N-methylmorpholine,N-methylpiperidine, preferably, the reaction is carried out in thepresence of diisopropylethylamine.

These steps, (i) and (ii), may be repeated until the formation of thedesired decapeptide.

To achieve the desired sequence of amino acids resulting in degarelix inthe second step, the following terminally protected amino acids need tobe coupled in a sequential order: Pg-Ser-OH, Pg-3-Pal-OH, Pg-D-Cpa-OHand Pg-D-Nal-OH in stepwise manner. The amino acid residue can beprotected with a side chain protecting group, if required. PreferablyPg-Ser(Z)-OH is used in this peptide synthesis, wherein Z is a sidechain protecting group, preferably a t-butyl protecting group.

In step d), after the desired amino acid sequence has been completed,the terminal protecting group is removed by treatment with a base,preferably selected from the group consisting of piperidine,triethylamine, DBU, N-methylmorpholine and N-methylpiperidine, mostpreferably with piperidine.

In step e), after deblocking the terminal amino group and while desiredside chain groups remain protected, acetylation is carried out.Preferably, this reaction can be carried out with acetic acid, in thepresence of diisopropyl or dicyclohexyl carbodiimide (DIC or DCC), or bysome other suitable acylation reaction, for example with acetylimidazole. Alternatively, acetylation is carried out by reacting thepeptide with acetic anhydride.

In step f), the acetylated decapeptide is decoupled from the resin bytreating the coupled decapeptide with an acid, preferably withtrifluoroacetic acid, which not only cleaves the peptide from the resin,but also cleaves all remaining side chain protecting groups to givedegarelix acetate.

A preferred method for the synthesis of degarelix using the abovedescribed synthesis based on the use of a compound of formula II isdescribed in FIG. 1. FIG. 1 also illustrates one way to achieve acompound of formula II, involving the use of a compound of formula VII.

Another synthetic approach describing the conversion process of acompound of formula II into degarelix or a pharmaceutically acceptablesalt thereof, —which is based on incorporation of the p-nitrophenylalanine residues at the 5^(th) position in the decapeptidesequence—is characterized by continuing with a solid phase synthesisthereby adding all the amino acids required for the decapeptide,preferably for degarelix. Only after completion of the peptidesynthesis, the nitro group of the amino acid residue at the 5^(th)position is reduced to an amino group and further modified to Aph(Hor).

The process comprises the steps of:

-   -   a) providing the compound of formula II,    -   b) repeating the following steps (i)-(ii) until a protected        decapeptide attached to the resin is formed:        -   (i) deprotecting the protected peptide attached to the resin            to remove the terminal protecting group, (        -   ii) coupling of another amino acid (according to the            required sequence), which is protected at the N-terminus to            the terminal amino group residue attached to the resin using            a coupling reagent to form protected peptide attached to the            resin, that is extended by one amino acid;    -   c) deprotecting the protected decapeptide attached to resin to        remove the terminal protecting group,    -   d) acetylating the N-terminus of the resulting decapeptide in        the presence of an acetylating agent to give the compound of        formula V,    -   e) treating the compound of formula V with a reducing agent to        form the compound of formula VI,    -   f) reacting the compound of formula VI with dihydroorotic acid        in the presence of a coupling reagent to form the protected        decapeptide attached to the resin; and    -   g) cleaving the decapeptide from the resin to form degarelix or        a pharmaceutically acceptable salt thereof.

In step b), the compound of formula II is converted to a protecteddecapeptide attached to the resin by usual solid phase peptidesynthesis, which involves first removing the terminal protecting groupand then coupling with terminally protected amino acids stepwise in thedesired order until the decapeptide is formed.

In a first step (i), the N-terminal protecting group—if it is Fmoc—canbe removed by treatment with a base, preferably an organic base, morepreferably an amine. The base may be selected from the group consistingof piperidine, piperazine, DBU and diethylamine, preferably piperidineand DBU, and most preferably, piperidine are used.

In a second step (ii), coupling of the protected peptide bound to theresin with the next protected amino acid is carried out in the presenceof a coupling reagent selected from the group comprising of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide,2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), or any coupling reagents on the base of uronium or phosphoniumsalts. Preferably, the reaction is carried out in the presence of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate.

The coupling reaction may be carried out in the presence of a baseselected from the group comprising of tertiary amine likediisopropylethylamine, triethylamine, N-methylmorpho line,N-methylpiperidine, preferably, the reaction is carried out in thepresence of diisopropylethylamine.

The first and the second step (i) and (ii) may be repeated until thedesired decapeptide is formed.

To produce degarelix as the preferred decapeptide, described in thesecond step the following terminally protected amino acids have to becoupled sequentially in stepwise manner: Pg-Ser-OH, Pg-3-Pal-OH,Pg-D-Cpa-OH and Pg-D-Nal-OH. The amino acid residue can be protectedwith a side chain protecting group, if required. Preferably Pg-Ser(Z)—OHis used for the peptide synthesis, wherein Z is side chain protectinggroup. Preferably Z is a hydroxy protecting group such as, for example,tBu or trityl group.

After the desired amino acid sequence has been completed, in step c) theterminal protecting group is removed by treatment with a suitable base,according to the conditions as described above, preferably selected fromthe group consisting of piperidine, triethylamine, DBU,N-methylmorpholine and N-methylpiperidine, most preferably piperidine.

In step d), after deblocking the terminal amino group and while desiredside chain groups remain protected, acetylation is preferably carriedout. Preferably, the resin bound peptide is treated with acetic acid, inthe presence of diisopropyl or dicyclohexyl carbodiimide (DIC or DCC).Alternatively the reaction can be carried out with acetic anhydride, orby some other suitable acylation reaction, for example with acetylimidazole. The acetylation process results in the compound of formula V.

In step e) the compound of formula V is reduced to compound of formulaVI. The reducing agent used is selected from, but not limited to, sodiumdithionite or tin (II) chloride. Preferably, the reduction is carriedout in the presence of tin (II) chloride.

In step f) the compound of formula VI is reacted with activateddihydroorotic acid, to form a protected decapeptide attached to theresin. If the activation is based on the presence of a coupling agent,the coupling reagent may be selected from the group comprisingN,N′-diisopropylcarbodiimide, dicyclohexylcarbodiimide andethyl-dimethyl-aminopropyl carbodiimide (EDC). Preferably, the reactionis carried out in the presence N,N′-diisopropylcarbodiimide.

The reaction may also be carried out in the presence of a couplingreagent and an additive. The addition of an additive has been found toreduce racemisation during the formation of amide linkage (i.e. peptidebond formation). An additive, when added to the coupling reactionresults in an increased yield and reduced racemisation rates of thepeptides formed. Additives form activated esters with amino acids. Theysuppress the side-reaction, such as formation of oxazolone andN-acylurea. A suitable additive is preferably selected from the groupcomprising of 1-hydroxybenzotriazo le (HOBt), 2-hydroxypyridine N-oxide,N-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole andendo-N-hydroxy-5-norbornene-2,3-dicarboxamide. More preferably, thereaction is carried out in the presence of N,N′-diisopropylcarbodiimideand 1-hydroxybenzotriazole.

In step g), the desired peptide is decoupled from the resin by treatingthe resin bound peptide with an acid, preferably trifluoroacetic acid,which not only cleaves the peptide from the resin, but also cleaves allremaining side chain protecting groups.

A preferred method for the synthesis of degarelix using the abovedescribed synthesis, based on the use of a compound of formula II isillustrated in FIG. 2. FIG. 2 also illustrates an alternative way tosynthesize the compound of formula II.

Degarelix prepared by the process(es) of the present invention ischaracterized by an impurity profile which allows for a more effectivepurification via the standard purification method, HPLC purification.Moreover, it allows preparing degarelix without previous synthesis ofAph derivatives, thereby significantly simplifying the syntheticapproach and allowing for a more economic process.

The compound of formula II as used in the processes described above canbe prepared by the novel processes described below.

In another aspect, a process for the solid phase synthesis of thecompound of formula II is provided, comprising the steps of:

a) reducing the compound of formula VII with a reducing agent to formthe compound of formula VIII,

b) reacting the compound of formula VIII with alkyl isocyanate or alkalimetal cyanate to form the compound of formula IX, and

c) deprotecting the compound of formula IX to remove the terminalprotecting group followed by coupling with a protected amino acid offormula X in the presence of a coupling reagent to form the compound offormula II

In step a) the compound of formula VII is reduced to a compound offormula VIII. The reducing agent used is selected from, but not limitedto, sodium dithionite or tin (II) chloride. Preferably, the reduction iscarried out in the presence of tin (II) chloride.

In step b), the reaction of the compound of formula VIII with alkalimetal cyanate, preferably with potassium cyanate or sodium cyanateresults in the compound of formula IX, wherein the side chain protectinggroup Y is hydrogen. In another alternative way, the reaction of thecompound of formula VIII with alkyl isocyanate, such as tert-butylisocyanate or trityl isocyanate results in the compound of formula IX,wherein the side chain protecting group Y is alkyl. The reaction may becarried out in the solvents selected from the group comprising of polaraprotic solvents like dimethylformamide, or N-methylpirrolidone.Preferably, the reaction is carried out in dimethylformamide.

In step c), the N-terminal protecting group—if it is Fmoc—can be removedby treatment with a base, preferably an organic base, more preferably anamine. The base may be selected from the group consisting of piperidine,piperazine, DBU and diethylamine, preferably piperidine and DBU, andmost preferably, piperidine are used. After removal of the protectinggroup, the compound of the resulting peptide is coupled with a compoundof formula X (L isomer) in the presence of a coupling agent to give thecompound of formula II. The coupling reagent can be selected from thegroup comprising of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide,2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) or any coupling reagents on the basis of uronium or phosphoniumsalts. Preferably, the reaction is carried out in the presence of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate.

In another aspect, the compounds of formula II, V and VII are providedas novel intermediates for the synthesis of decapepeptides, preferablyfor the synthesis of degarelix. These intermediate peptides aredisclosed herewith in their free form (where Resin is H), as well as intheir form bound to a solid support (where Resin is a solid support).

The compound of formula VII, which is employed in a process to prepare acompound of formula II is novel and can be prepared as described below.

In still another aspect, a process for the synthesis of the compound offormula VII is provided characterized as comprising the steps ofdeprotecting the compound of formula XI to remove the terminalprotecting group followed by coupling with a protected amino acid offormula X (D-isomer) in the presence of a coupling reagent to form thecompound of formula VII.

The terminal protecting group can be removed by treatment with a base,preferably an organic base, more preferably an amine. The base may beselected from the group consisting of piperidine, piperazine, DBU anddiethylamine, preferably piperidine and DBU, and most preferably,piperidine are used. After the removal of the protecting group, theresulting peptide is coupled with the D-isomer of the compound offormula X. The coupling is performed in the presence of a coupling agentand results in the formation of the compound of formula VII. Thecoupling reagent can be selected from the group comprising of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide,2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate(HBTU) or any coupling reagents on the base of uronium or phosphoniumsalts. Preferably, the reaction is carried out in the presence of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate.

The solid phase synthesis as described above may be carried out inaprotic solvents selected from the group comprising dimethylformamide,and N-methylpyrrolidone. Preferably, the reaction is carried out indimethylformamide.

A preferred method for releasing degarelix from the solid support is byacidic treatment, more preferably, by treatment with TFA.

By using the synthetic approaches described above the incorporation ofthe entire residues Aph(Hor) and D-Aph(Cbm), or at least theincorporation of Aph(Hor) can be avoided. Thereby the complicationsaccompanying the insertion of such a bulky amino acid residue can beeliminated, and the formation of corresponding des-peptides (deletionpeptides, which lack one or more amino acids) is strongly decreased.Therefore, the construction of Aph(Hor)- or Aph (Hor)- andD-Aph(Cbm)-group in the peptide can noticeably enhance the purity of theobtained crude peptide, in particular when performed as solid statepeptide synthesis. The impurity profile that can be achieved with thisnew synthesis is favorable for the routine follow-up purification withHPLC because the impurities are easier to separate. Moreover, the methodcan noticeably facilitate the overall synthesis of the peptide, since inthis case there is no need to prepare and purify the intermediate Aphderivatives to be inserted into peptide sequence. All the side productsand excess of the reagents during the construction of these residues canbe easily removed by filtration and washing.

Thus, the synthetic approaches described above employ less bulkyintermediates, by using the compounds of Formula II and/or X, avoid theuse of hazardous reagents and provide a simple process for the synthesisof degarelix or salts thereof, which results in a product with higheryield and/or higher purity than the methods known in the art.

Preferred Embodiments of the Invention

-   -   1. A method for preparing a peptide, wherein the peptide is        characterized as being a peptide with at least one Aph        derivative, preferably selected from Aph(Hor) and Aph(Cbm), in        the peptide sequence or a pharmaceutically acceptable salt        thereof, wherein the method comprises the use of either        p-nitrophenylalanine or a nitro-peptide, preferably resulting in        incorporation of at least one or preferably one        nitrophenylalanine into the peptide sequence; followed by        modification of incorporated nitrophenylalanine residues within        the peptide sequence into the respective Aph derivative.        Preferably the peptide is a decapeptide. More preferably        Aph(Hor) and/or Aph(Cbm) are in the 5^(th) and 6^(th) position.    -   2. A method according to embodiment 1 wherein the peptide is        degarelix of formula I, or a pharmaceutically acceptable salt        thereof.    -   3. A method according to embodiment 1 or 2 wherein said        nitro-peptide is characterized as comprising one or two        p-nitrophenylalanine residues, and preferably wherein said        nitro-peptide is a compound of formula II,V or VII, most        preferably of formula II    -   4. A method according to any of the embodiments above, wherein        the method comprises converting a compound of formula II,        wherein Pg is a terminal protecting group, X and Y are side        chain protecting groups, and Resin is H or a solid support, into        degarelix or a pharmaceutically acceptable salt thereof.    -   5. A method according to any of the embodiments above, wherein        the method is performed as solid phase peptide synthesis.

6. The method according to any of the embodiments above, characterizedas comprising the steps of:

-   -   a) treating the compound formula II with a reducing agent to        form a compound of formula III, wherein Pg is a terminal        protecting group, X and Y are side chain protecting groups, and        Resin is a solid support    -   b) reacting the compound of formula III with an activated        dihydroorotic acid to form a compound formula IV, wherein Pg is        a terminal protecting group, X and Y are side chain protecting        groups, and Resin is a solid support    -   c) repeating the following steps (i)-(ii) attached to resin        until a protected decapeptide is formed:        -   (i) deprotecting the protected peptide to remove the            terminal protecting group,        -   (ii) coupling of the protected amino acid (according to            required sequence) to the terminal amino group residue of            the peptide attached to the resin using a coupling reagent            to form a by one amino acid elongated protected peptide,    -   d) deprotecting the protected decapeptide to remove the terminal        protecting group,    -   e) acetylating the N-terminus of the resulting decapeptide in        the presence of an acetylating agent, and    -   f) cleaving the acetylated decapeptide peptide from the resin to        form degarelix or a pharmaceutically acceptable salt thereof;        -   thereby converting a compound of formula II in degarelix or            a pharmaceutically acceptable salt thereof.

7. The method according to embodiment 6, wherein in step b) theactivated dihydroorotic acid is activated by a coupling reagent, whichis selected from a group comprising N,N′-diisopropylcarbodiimide,dicyclohexylcarbodiimide and ethyl-dimethyl-aminopropyl carbodiimide(EDC), preferably said coupling reagent is N,N′-diisopropylcarbodiimide,preferably N,N′-diisopropylcarbodiimide in combination with1-hydroxybenzotriazo le.

8. The method according to any of embodiments 1 to 5, comprising thesteps of:

-   -   a) providing the compound of formula II,    -   b) repeating the following steps (i)-(ii) until a protected        decapeptide attached to resin is formed:        -   (i) deprotecting the protected peptide attached to the resin            to remove the terminal protecting group,        -   (ii) coupling of the protected amino acid according to            required sequence to the terminal amino group residue            attached to the resin using a coupling reagent to form            protected peptide attached to the resin;    -   c) deprotecting the protected decapeptide to remove terminal        protecting group,    -   d) acetylating the N-terminus of the resulting decapeptide in        the presence of an acetylating agent to result in a compound of        formula V, wherein X, Y and Z are side chain protecting groups,        and Resin is a solid support;    -   e) treating the compound of formula V with a reducing agent to        form a compound of formula VI, wherein X, Y and Z are side chain        protecting groups, and Resin is a solid support;    -   f) reacting the compound of formula VI with activated        dihydroorotic acid to form a protected decapeptide attached to        the resin; and    -   g) cleaving the decapeptide from the resin to form degarelix or        a pharmaceutically acceptable salt thereof.

9. The method according to embodiment 8, wherein in step e) the reducingagent is selected from sodium dithionite or tin (II) chloride,preferably tin (II) chloride.

10. The method according to embodiment 8, wherein in step f) theactivated dihydroorotic acid is activated by a coupling reagent, whichis selected from the group comprising N,N′-diisopropylcarbodiimide,dicyclohexylcarbodiimide and ethyl-dimethyl-aminopropyl carbodiimide(EDC), preferably said coupling reagent is N,N′-diisopropylcarbodiimide,more preferably N,N′-diisopropylcarbodiimide in combination with1-hydroxybenzotriazole.

11. The compound of formula II, wherein Pg is a terminal protectinggroup or H, X and Y are side chain protecting groups or H, and Resin isa solid support or, and wherein the compound is not attached to a solidsupport, the Resin is H.

12. A method for preparing the compound of formula II, characterized ascomprising the following steps:

-   -   a) reducing a compound of formula VII, wherein Pg is a terminal        protecting group; X is a side chain protecting group, and Resin        is a solid support with a reducing agent to form a compound of        formula VIII, wherein Pg is a terminal protecting group; X is a        side chain protecting group, and Resin is a solid support;    -   b) reacting the compound of formula VIII with alkyl isocyanate        or alkali metal cyanate to form a compound of formula IX,        wherein Pg is a terminal protecting group, X and Y are side        chain protecting groups, wherein Y is H when an alkali metal        cyanate is present, and Y is tBu or another alkyl group, when        alkyl isocyanate is present and Resin is a solid support;    -   c) deprotecting the compound of formula IX to remove the        terminal protecting group followed by coupling with the        protected D-isomer of the amino acid of formula X, wherein Pg is        a terminal protecting group, preferably Fmoc, in the presence of        a coupling reagent to form the compound of formula II.

13. The process according to embodiment 6 or 12, wherein in step a) thereducing agent is sodium dithionite or tin (II) chloride, preferably tin(II) chloride.

14. The method according to embodiment 12, wherein preparing thecompound of formula VII, comprises the step of deprotecting the compoundof formula XI, wherein Pg is a terminal protecting group, X is a sidechain protecting group and Resin is a solid support; by removing theterminal protecting group; followed by a step of coupling the D isomerof the protected amino acid of formula X in the presence of a couplingreagent to form the compound of formula VII.

15. The process according to embodiment 12, wherein the coupling reagentis selected from the group consisting of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide and2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), preferably, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU).

16. A compound of formula V, wherein X, Y and Z are side chainprotecting groups, or H and Resin is a solid support and, wherein thecompound is not attached to a solid support, the Resin is H.

17. A compound of formula VII, wherein Pg is a terminal protecting groupor H, X is a side chain protecting group or H and Resin is a solidsupport and/or, wherein the peptide is not attached to a solid supportthe Resin is H.

18. Use of a compound of formula X, formula II, formula V or of formulaVII for the synthesis of a peptide, preferably for synthesis ofdegarelix.

19. A method according to any of the embodiments describing methods ofsynthetic processes above characterized as being based on the Fmocprotection strategy, wherein the terminal protection group Pg is thebase-labile protective group Fmoc.

Abbreviations

Aph Aminophenylalanin Amf Aminomethylphenylalanine For Formyl Imz2-imidazolidone-4-carbonyl GnRH Gonadotropin releasing hormone SPPSSolid phase peptide synthesis LPPS Liquid phase peptide synthesis MBHAresin Methyl benzhydryl amide resin Fmoc-Rink amide AM-resin4-(2′,4′-Dimethoxyphenyl-Fmoc- aminomethyl)-phenoxyacetamidomethylpolystyrene resin Fmoc-Rink amide-MBHA resin4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-4-methylbenzhydrylamine polystyrene resinFmoc-D-Ala-Rink resin 9-Fluorenylmethyloxycarbonyl-D-alanine-Rink resinFmoc-D-Ala-OH 9-Fluorenylmethyloxycarbonyl-D-alanine Fmoc-Pro-OH9-Fluorenylmethyloxycarbonyl-L-proline Fmoc-Lys(iPr, Boc)-OH9-Fluorenylmethyloxycarbonyl-N(ε)-isopropyl- N(ε)-Boc-lysine Fmoc-Leu-OH9-Fluorenylmethyloxycarbonyl-leucine-OH Fmoc-D-Phe(NO₂)-OHFluorenylmethoxycarbonyl-D-4- nitrophenylalanine Fmoc-Phe(NO₂)-OHFluorenylmethoxycarbonyl-4-L- nitrophenylalanine Fmoc-D-Aph(Cbm)-OH9-Fluorenylmethyloxycarbonyl-N(4)-carbamoyl- D-4-aminophenylalanineFmoc-Ser(tBu)-OH 9-Fluorenylmethyloxycarbonyl-O-t-butyl-serineFmoc-D-3-Pal-OH 9-Fluorenylmethyloxycarbonyl-D-3- pyridylalanineFmoc-D-Cpa-OH/Fmoc-D-Phe(4-Cl)-OH 9-Fluorenylmethyloxycarbonyl-D-4-chlorophenylalanine Fmoc-D-Nal-OH 9-Fluorenylmethyloxycarbonyl-D-2-naphtylalanine Fmoc-Aph(L-Hor)-OH 9-Fluorenylmethyloxycarbonyl-N(4)-(L-hydroorotyl)-4-aminophenylalanine Aph(Hor)N(4)-(L-hydroorotyl)-4-aminophenylalanine D-Aph(Cbm)4-(Aminocarbonyl)amino-D-Phenylalanine Aph(Trt)4-(trityl)amino-D-Phenylalanine Hor Dihydroorotyl Fmoc9-Fluorenylmethyloxycarbonyl Boc t-Butyloxycarbonyl Dde1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene HPLC High pressure liquidchromatography L-p-nitro-Phe L-p-nitrophenylalanine D-p-nitro-PheD-p-nitrophenylalanine DIPEA Diisopropylethylamine tBu-NCO tert-butylisocyanate Ac₂O Acetic anhydride SnCl₂ Tin (II) chloride Hor-OHDihydroorotic acid HOBt 1-Hydroxybenzotriazole. TFA Trifluoroacetic acidDCC N,N′-dicyclohexylcarbodiimide EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide DIC Diisopropylcarbodiimide HBTU2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate

EXAMPLES

Detailed experimental parameters suitable for the preparation ofdegarelix according to the present invention are provided by thefollowing examples, which are intended to be illustrative and notlimiting of all possible embodiments of the invention.

Example 1 Preparation of Degarelix Via Solid Phase Synthesis Step 1:Preparation of Fmoc-D-Phe(p-NO₂)-Leu-Lys(Boc, iPr)-Pro-D-Ala-Rink Resin

Synthesis of the protected peptide was carried out by step-by-step solidphase peptide synthesis using Rink amide resin (200 mg, loading 0.65mmol/g). After swelling of the resin in 2 ml of dimethylformamide Fmocprotective group was removed by 20% solution of piperidine indimethylformamide (2×2 ml, 5 min and 20 min) and the resin was washedwith dimethylformamide (4×2 ml). Fmoc-D-Ala-OH, Fmoc-Pro-OH,Fmoc-Lys(Boc, iPr)—OH, Fmoc-Leu-OH and Fmoc-D-Phe(p-NO₂)—OH (three-foldexcess respect to the loading of the resin and two-fold excess in caseof Fmoc-Lys(Boc, iPr)—OH) were activated by HBTU (150 mg, 0.4 mmol) inpresence of diisopropylethylamine (136 μl, 0.67 mmol) and coupled to theresin in 50 min to get Fmoc-protected pentapeptide. The intermediateFmoc deprotection was carried out as described above.

Step 2: Preparation of Fmoc-D-Aph-Leu-Lys(Boc, iPr)-Pro-D-Ala-Rink Resin

The obtained peptide resin was treated with a solution of SnCl₂ (0.6 g,3.2 mmol) and diisopropylethylamine (70 μl, 0.4 mmol) in 2.5 ml ofdimethylformamide for 15 hours. At the end of the reaction the solventwas filtered off and the resin was washed with dimethylformamide (3×2ml).

Step 3: Preparation of Fmoc-D-Aph(Cbm)-Leu-Lys(Boc, iPr)-Pro-D-Ala-RinkResin

A solution of potassium cyanate (60 mg, 0.74 mmol) in 2.5 ml ofdimethylformamide, containing 10% of 85% phosphoric acid (v/v) was addedto the obtained peptide linked to the resin. The reaction was left tocontinue for 6 hours, the solvent was filtered off and the freshlyprepared solution of potassium cyanate was added. After 6 hours theresin was washed with dimethylformamide (3×2 ml), Fmoc protective groupwas removed by 20% solution of piperidine in dimethylformamide (2×2 ml,5 min and 20 min) and the resin was washed again with dimethylformamide(4×2 ml).

Step 4: Preparation of Fmoc-Aph-D-Aph(Cbm)-Leu-Lys(Boc,iPr)-Pro-D-Ala-Rink Resin

Fmoc-Phe(p-NO₂)-OH (170 mg, 0.4 mmol) was activated with HBTU (150 mg,0.4 mmol) in presence of diisopropylethylamine (136 μl, 0.67 mmol) andadded to the peptide resin. After completion of the coupling reactionthe resin was washed with dimethylformamide (3×2 ml) and treated with asolution of SnCl₂ (0.6 g, 3.2 mmol) and diisopropylethylamine (70 μl,0.4 mmol) in 2.5 ml of dimethylformamide for 15 hours. At the end of thereaction the resin was washed with dimethylformamide (3×2 ml).

Step 5: Preparation of Fmoc-Aph(Hor)-D-Aph(Cbm)-Leu-Lys(Boc,iPr)-Pro-D-Ala-Rink Resin

A solution of dihydroorotic acid (60 mg, 0.36 mmol),diisopropylcarbodiimide (60 μl, 0.39 mmol), hydroxybenzotriazole (52 mg,0.39 mmol) and diisopropylethylamine (114 μl, 0.65 mmol) in 2.5 ml ofdimethylformamide was added to the resin. After 2 and 4 hours thesolvent was filtered off and freshly prepared mixture of dihydrooroticacid, N,N-diisopropylcarbodiimide, hydroxybenzotriazole anddiisopropylethylamine was added. Then the peptide resin was washed withdimethylformamide (3×2 ml) and Fmoc protective group was removed by 20%solution of piperidine in dimethylformamide (2×2 ml, 5 min and 20 min)followed by washing with dimethylformamide (4×2 ml).

Step 6: Preparation ofAc-D-Nal-D-Cpa-D-Pal-Ser(tBu)-Aph(Hor)-D-Aph(Cbm)-Leu-Lys(iPr,Boc)-Pro-D-Ala-Rink Resin

Fmoc-Ser(tBu)-OH, Fmoc-D-Pal-OH, Fmoc-D-Cpa-OH and Fmoc-D-Nal-OH(three-fold excess respect to the loading of the resin) were activatedby HBTU (150 mg, 0.4 mmol) in presence of diisopropylethylamine (136 μl,0.67 mmol) and coupled to the resin in 50 min to get Fmoc-protecteddecapeptide. The intermediate Fmoc deprotections were carried out using20% solution of piperidine in dimethylformamide (2×2 ml, 5 min and 20min) followed by washing with dimethylformamide (4×2 ml). Uponcompletion of the synthesis N-terminal amino group was acetylated withthe mixture of acetic acid (23 μl, 0.4 mmol), diisopropylcarbodiimide(60 μl, 0.39 mmol), hydroxybenzotriazole (52 mg, 0.39 mmol) anddiisopropylethylamine (114 μl, 0.65 mmol), peptide resin was washed withdimethylformamide (2×2 ml), dichloromethane (2×2 ml) and dried.

Step 7: Cleavage of Degarelix From the Resin

Dry peptide resin was suspended in 4 ml of the mixture of 1% water intrifluoroacetic acid and stirred for 1.5 hours. Then the resin wasfiltered and washed with 1 ml of trifluoroacetic acid. The organicsolutions were collected and dried in vacuo. The solid residue waswashed with methyl t-butyl ether and dried to get crude degarelix withoverall yield 100 mg (50%) and HPLC purity 70%.

Example 2 Preparation of Degarelix Via Solid Phase Synthesis Step 1:Preparation ofAc-D-Nal-D-Cpa-D-Pal-Ser(tBu)-Phe(p-NO₂)-D-Aph(Cbm)-Leu-Lys(iPr,Boc)-Pro-D-Ala-Rink Resin

Synthesis of the protected peptide was carried out by step-by-step solidphase peptide synthesis using Rink amide resin (3 g, loading 0.65mmol/g). After swelling of the resin in 12 ml of dimethylformamide Fmocprotective group was removed by 20% solution of piperidine indimethylformamide (2×12 ml, 5 min and 15 min) and the resin was washedwith dimethylformamide (4×12 ml). Fmoc-D-Ala-OH, Fmoc-Pro-OH,Fmoc-Lys(iPr, Boc)-OH, Fmoc-Leu-OH, Fmo c-D-Aph(Cbm)-OH,Fmoc-Phe(p-NO₂), Fmoc-Ser(tBu)-OH, Fmoc-D-Pal-OH, Fmoc-D-Cpa-OH,Fmoc-D-Nal-OH (three-fold excess respect to the loading of the resin andtwo-fold excess in case of Fmoc-Lys(iPr, Boc)-OH) were activated by HBTU(2.2 g, 5.8 mmol) in presence of diisopropylethylamine (1.7 ml, 9.8mmol) and coupled to the resin in 60 min. The solution was filtered offand the resin was washed with dimethylformamide (3×12 ml). After eachcoupling the unreacted amino groups, as well as the N-terminal aminogroup, were capped using a solution of acetic anhydride (0.54 ml) anddiisopropylethylamine (0.93 ml) in 10.5 ml of dimethylformamide.

Step 2: Preparation ofAc-D-Nal-D-Cpa-D-Pal-Ser(tBu)-Aph-D-Aph(Cbm)-Leu-Lys(iPr,Boc)-Pro-D-Ala-Rink resin

The obtained peptide resin was treated with a solution of tin (II)chloride dihydrate (4.4 g, 0.019 mol) and diisopropylethylamine (0.4 ml,2.3 mmol) in 12 ml of dimethylformamide for 10 hours. At the end of thereaction the solvent was filtered off and the resin was washed withdimethylformamide (3×12 ml).

Step 3: Preparation ofAc-D-Nal-D-Cpa-D-Pal-Ser(tBu)-Aph(Hor)-D-Aph(Cbm)-Leu-Lys(iPr,Boc)-Pro-D-Ala-Rink Resin

A solution of dihydroorotic acid (0.62 g, 3.9 mmol),diisopropylcarbodiimide (0.6 ml, 3.9 mmol), 1-hydroxybenzotriazole (0.53g, 3.9 mmol) in 18 ml of dimethylformamide was stirred for 30 min andadded to the peptide resin. After 1.5 hours the solution was filteredoff and a freshly prepared mixture of dihydroorotic acid,N,N-diisopropylcarbodiimide, hydroxybenzotriazole anddiisopropylethylamine was added. After 1.5 hours the peptide resin waswashed with dimethylformamide (3×12 ml), dichloromethane (3×12 ml) anddried in vacuo.

Step 4: Cleavage of the Peptide From the Resin

Dry peptide resin (5 g) was suspended in 30 ml of a mixture of 5% waterand 95% trifluoroacetic acid and stirred for 1.5 hours. Then the resinwas filtered off and the solution was stirred for 1.5 hours more. Methyltert-buthyl ether (100 ml) cooled to 4° C. was added to the solution ofthe peptide and stirred for 90 min. The procedure was repeated againwith the resin remained after the first cleavage. The combinedsuspensions were filtered and the peptide was dried in vacuo to getcrude degarelix with overall yield 55% and HPLC purity 84%.

Example 3 Reduction of Fmoc-Phe(NO2)-Leu-Lys(iPr)-Pro-Ala-Resin 3.1.With Sodium Dithionite (DEG-001-18)

Stock solution for the reduction: Na₂S20₄ 1050 mg (5 mmol), K₂CO₃ 968 mg(7 mmol), TBAHS 170 mg (0.5 mmol) in 5 ml of water (Ref.: TetrahedronLett. 54 (2013) 2600-2603).

130 mg of peptidyl-resin (swelled in DCM)+0.5 ml of DCM+0.5 mL of stocksolution, reaction 2 h, washings 3×1 ml of DCM/water (v/v 1:1), DMF,MeOH and DCM.

Cleavage with the mixture TFA/water 99/1.

Product: Fmoc-4-Aph-Leu-Lys(iPr)-Pro-Ala-NH2 (M=810)

Analytical Method

Instrument Agilent UPLC 1290 series + Agilent 6530 mass accuracy Q-TOFColumn C8 Zorbax Eclipse plus Agilent 07/13R 4.6 × 50 mm 1.8-Micron PN9599941-906 Mobile phase A (FM A) water + 0.1% TFA Mobile phase B (FM B)ACN + 0.07% TFA Flow rate  1 ml/min Detection 226 nm Run time  40 minColumn temperature 30° C. Gradient elution 0 min-5% FM B, 1 min-5% FM B,31 min-90% FM B, 36 min-90% FM B, 37 min-5%, 40 min-5%

HPLC Result as Illustrated in FIG. 3

Peak RT RRT Area % 1 3.20 0.29 2.13 2 3.37 0.30 12.08 3 8.07 0.72 7.16 411.22 1.00 57.52 5 11.42 1.02 0.43 6 11.69 1.04 6.67 7 12.05 1.07 0.92 812.22 1.09 1.25 9 13.07 1.16 0.61 10 24.09 2.15 2.53

Main peak (product): RT=11.22 min, purity 53.24%, [M+H]+=811.45

Main impurities: RT=3.15 min, [M+H]+=669.30 (−142, not identified)

-   -   RT=3.37, 11.10%, [M+H]+=589.38 (−222, -Fmoc)    -   RT=8.07, 5.30%, no TIC signal    -   RT =11.69, 6.13%, [M+H]+=891.40 (+90, not identified)

Results: The reduction is completed in 2 h, but partialFmoc-deprotection occurs (17% respect to the quantity of the product)

3.2 With Tin(II)Chloride (DEG-001-35-2)

130 mg of peptidyl-resin was treated with a solution of tin (II)chloride dehydrate (0.19 g, 0.82 mmol) and diisopropylethylamine (30 μl,0.16 mmol) in 2 ml of dimethylformamide for 10 hours. At the end of thereaction the solvent was filtered off and the resin was washed withdimethylformamide (2×2 ml) and DCM (2×2 ml).

Cleavage with the mixture TFA/water 99/1.

Product: Fmoc-4-Aph-Leu-Lys(iPr)-Pro-Ala-NH2 (M=810)

HPLC Result as Illustrated in FIG. 4

No Fmoc-deprotected product was observed*

*the peak with rt 8.07 was seen in both cases. It do not give a TICsignal, probably, it is not a peptide impurity (salts or non-peptideorganic compound). It was not taken into consideration while calculatingHPLC purity.

Comparative Table

Method of reduction HPLC purity, % Fmoc-deprotected product, % Sodiumdithionite 65 17 Tin (II) chloride 67 None

1. A method for preparing a decapeptide, wherein the decapeptide ischaracterized as being a peptide with at least one Aph derivative, inthe 5th positon and one Aph derivative in the 6th position in thedecapeptide sequence, wherein the Aph derivative is selected fromAph(Hor) and Aph(Cbm), or a pharmaceutically acceptable salt thereof,wherein the method comprises the use of either p-nitrophenylalanine or acompound of formula X,

wherein Pg is a terminal protecting group; or a nitro-peptide, which ischaracterized as a peptide comprising one or two p-nitrophenylalanineresidues, followed by modification of nitrophenylalanine residuesincorporated in the peptide sequence into the respective Aph derivativeAph(Hor) or Aph(Cbm), wherein the decapeptide is degarelix of formula I,

or a pharmaceutically acceptable salt thereof.
 2. A method according toclaim 1 wherein said nitro-peptide is a compound of formula II, V orVII.
 3. A method according to claim 2 any of the claims above, whereinthe method comprises converting a compound of formula II,

wherein Pg is a terminal protecting group, X and Y are side chainprotecting groups, and Resin is H or a solid support, into degarelix ora pharmaceutically acceptable salt thereof.
 4. A method according toclaim 1, wherein the method is performed as solid phase peptidesynthesis.
 5. The method according to claim 2, further comprising thesteps of: a) treating the compound of formula II with a reducing agentto form a compound of formula III,

wherein Pg is a terminal protecting group, X and Y are side chainprotecting groups, and Resin is a solid support; b) reacting thecompound of formula III with an activated dihydroorotic acid to form acompound of formula IV,

wherein Pg is a terminal protecting group, X and Y are side chainprotecting groups, and Resin is a solid support; c) repeating thefollowing steps (i)-(ii) attached to resin until a protected decapeptideis formed: (i) deprotecting the protected peptide to remove the terminalprotecting group, (ii) coupling of the protected amino acid (accordingto required sequence) to the terminal amino group residue of the peptideattached to the resin using a coupling reagent to form a by one aminoacid elongated protected peptide, d) deprotecting the protecteddecapeptide to remove the terminal protecting group, e) acetylating theN-terminus of the resulting decapeptide in the presence of anacetylating agent, and f) cleaving the acetylated decapeptide from theresin to form degarelix or a pharmaceutically acceptable salt thereof;thereby converting a compound of formula II in degarelix or apharmaceutically acceptable salt thereof.
 6. The method according toclaim 2, comprising the steps of: a) providing the compound of formulaII, b) repeating the following steps (i)-(ii) until a protecteddecapeptide attached to resin is formed: (i) deprotecting the protectedpeptide attached to the resin to remove the terminal protecting group,(ii) coupling of the protected amino acid according to required sequenceto the terminal amino group residue attached to the resin using acoupling reagent to form protected peptide attached to the resin; c)deprotecting the protected decapeptide to remove terminal protectinggroup, d) acetylating the N-terminus of the resulting decapeptide in thepresence of an acetylating agent to result in a compound of formula V,

wherein X, Y and Z are side chain protecting groups, and Resin is asolid support; e) treating the compound of formula V with a reducingagent to form a compound of formula VI,

wherein X, Y and Z are side chain protecting groups, and Resin is asolid support f) reacting the compound of formula VI with activateddihydroorotic acid to form a protected decapeptide attached to theresin; and g) cleaving the decapeptide from the resin to form degarelixor a pharmaceutically acceptable salt thereof.
 7. A compound of formulaII

wherein Pg is a terminal protecting group or H, X and Y are side chainprotecting groups or H, and Resin is a solid support, and wherein thecompound is not attached to a solid support, the Resin is H.
 8. A methodfor preparing the compound of formula II of claim 7, comprising thefollowing steps: a) reducing a compound of formula VII,

wherein Pg is a terminal protecting group; X is a side chain protectinggroup, and Resin is a solid support with a reducing agent to form acompound of formula VIII,

wherein Pg is a terminal protecting group; X is a side chain protectinggroup, and Resin is a solid support; b) reacting the compound of formulaVIII with alkyl isocyanate or alkali metal cyanate to form a compound offormula IX, and

wherein Pg is a terminal protecting group, X and Y are side chainprotecting groups, wherein Y is H when an alkali metal cyanate ispresent, and Y is tBu or another alkyl group, when alkyl isocyanate ispresent and Resin is a solid support; c) deprotecting the compound offormula IX to remove the terminal protecting group followed by couplingwith the protected D isomer L isomer of the amino acid of formula X,

wherein Pg is a terminal protecting group, preferably Fmoc, in thepresence of a coupling reagent to form the compound of formula II. 9.The method according to claim 8, wherein preparing the compound offormula VII comprises the step of deprotecting the compound of formulaXI,

wherein Pg is a terminal protecting group, X is a side chain protectinggroup and Resin is a solid support; followed by a step of coupling the Disomer of the protected amino acid of formula X in the presence of acoupling reagent to form the compound of formula VII.
 10. A compound offormula V,

wherein X, Y and Z are side chain protecting groups or H, and Resin is asolid support and wherein the compound is not attached to a solidsupport the Resin is H.
 11. A compound of formula VII,

wherein Pg is a terminal protecting group or H, X is a side chainprotecting group or H, and Resin is a solid support and wherein thepeptide is not attached to a solid support the Resin is H.
 12. A methodof synthesizing degarelix comprising converting a compound of formulaII, a compound of formula V or a compound of formula VII.
 13. (canceled)14. The method according to claim 1, wherein the terminal protectiongroup Pg is the base-labile protective group Fmoc.
 15. The methodaccording to claim 8, wherein the terminal protection group Pg is thebase-labile protective group Fmoc.
 16. The method of claim 8, whereinsaid Pg group in step(c) is Fmoc.